Compositions and methods for the treatment of chronic lymphocytic leukemia

ABSTRACT

Methods for treating patients with CLL with pharmaceutical agents are disclosed. The methods of the present invention can be used in patients that have not responded to standard treatment. In addition, the methods can be used to augment the impact of standard chemotherapy.

This application is a 371 of PCT/US99/21518 filed Sep. 17, 1999 whichclaims benefit of Ser. No. 60/101,721 filed Sep. 24, 1998

FIELD OF THE INVENTION

The present invention pertains to the treatment of patients with chroniclymphocytic leukemia (CLL) with pharmaceutical compositions comprisingType 4 cyclic adenosine monophosphate phosphodiesterase inhibitors, andmore particularly, with an inhibitor that specifically inhibits Type 4cyclic adenosine monophosphate phosphodiesterase.

BACKGROUND

Leukemias are malignant neoplasms of hematopoietic tissues. Theseneoplasms are categorized into two predominant forms: chronic and acute.While acute leukemias are characterized by undifferentiated cellpopulations, chronic leukemias usually present a more mature morphology.Notwithstandmg these classifications, however, the pathologicalimpairment of normal hematopoiesis is the hallmark of all leukemias.

Chronic lymphocytic leukemia (CLL) is a neoplasm in which a clonalexpansion of small lymphocytes accumulate in the marrow, lymph nodes,blood, spleen liver, and sometime other organs. The CLL cell is theneoplastic counterpart of an immunologically immature, incompetentlymphocyte. In over 95 percent of the cases, the clonal expansion is ofa B-cell lineage. See Cancer: Principles & Practice of Oncology (3rdEdition) (1989) (pp. 1843-1847). In less than 5 percent of cases thetumor cells have a T-cell phenotype.

CLL, while accounting for only about 0.8 percent of all cancers in theUnited States, is the most prevalent leukemia afflicting adults inmodern. countries, accounting for 30 percent of all leukemias. Amajority of patients are over 60 years at the time of disease and 90percent are over age 50.

Most patients are diagnosed following a routine physical examination ora blood count. The earliest and most frequent symptoms are fatigue andmalaise. Later symptoms include lymphadenopathy and splenomegaly. Anemiaand thrombocytopenia is found in approximately 15 percent of patients.

The general goal of leukemia therapy is to arrest the proliferation ofabnormal morphologies and restore “normal” hematopoiesis in the bonemarrow. Treatment regimens include chemotherapy. Unfortunately,chemotherapy is not always successful. Indeed, while CLL patients mayhave initial clinical responses to alkylating agents such aschlorambucil or adenosine analogs such as fludarabine, many ultimatelybecome refractory to therapy. Consequently, there is a pressing need forthe identification of novel approaches to this disease.

SUMMARY OF THE INVENTION

The present invention pertains to the treatment of patients with chroniclymphocytic leukemia with pharmaceutical compositions comprising Type 4cyclic adenosine monophosphate phosphodiesterase inhibitors, and moreparticularly, with an inhibitor that specifically inhibits Type 4 cyclicadenosine monophosphate phosphodiesterase. One embodiment of the presentinvention contemplates a method comprising: a) providing: i) a patienthaving symptoms of chronic lymphocytic leukemia, and ii) a formulationcomprising an inhibitor that specifically inhibits Type 4 cyclicadenosine monophosphate phosphodiesterases; and b) administration of atherapeutically effective dose of said formulation to said patient underconditions such that said symptoms are reduced.

The present invention is not limited by the method of administration. Inone embodiment, the administration is enteral administration. In anotherembodiment, said enteral administration is oral administration. In stillanother embodiment, said administration is parenteral administration. Inthese embodiments, said parenteral administration can be topicaladministration or by a transdermal patch. In another embodiment, saidparenteral administration is subcutaneous administration. While in stillanother embodiment, said parenteral administration utilizes an aerosol.

The present invention is not limited by the nature of the patient. Inone embodiment, said patient is a naive patient (e.g., has not undergoneprior treatment for CLL), while in other embodiments said patient isunresponsive or refractory to standard chemotherapy (e.g., alkylatingagents). In still another embodiment, said patient is immunocompromised.In one embodiment, said patient is over fifty years of age.

The present invention is also not limited by the method of determiningresponse to treatment. In one embodiment, said symptoms compriselymphadenopathy and splenomegaly. In a yet another embodiment, saidsymptoms comprise the histology of a lymph node that is consistent withCLL.

The present invention contemplates usage of a variety of specificinhibitors. A preferred inhibitor is rolipram. While it is not intendedthat the present invention be limited to a specific mechanism by whichthe inhibitors of the present invention achieve therapeutic success, ithas been empirically found (as the data herein shows) that the specificinhibitors of the present invention (e.g., rolipram) augment apoptosisinduced by commonly used drugs (e.g., doxorubicin, chlorambucil andfludarabine). Consequently, the present invention specificallycontemplates the use of the inhibitors in combination with other drugs,including but not limited to cytotoxic drugs.

Definitions

As used herein, the term “enteral administration” means the introductionof a composition to a patient such that it is absorbed in the intestinaltract of the patient (e.g., pill, tablet, elixir, etc.)

As used herein, the term “oral administration” means the introduction ofa composition to a patient through the oral cavity (i.e., in the mouth).

As used herein, the term “parenteral administration” meansadministration of a composition other than enteral (e.g., injection,transdermal, aerosol, etc.).

As used herein, the term “topical administration” means the introductionof a composition to a patient by application to the surface of the skin.

As used herein, the term, “subcutaneous administration” meansintroduction of a composition to a patient under the surface of the skin(e.g., injection with a hypodermic needle).

As used herein, the phrase “naive patient” refers to a patient that hasnot undergone prior treatment for chronic lymphocytic leukemia.

As used herein, the phrase “an inhibitor that specifically inhibits Type4 cyclic adenosine monophosphate phosphodiesterase” refers to a compoundthat inhibits Type 4 but not Type 1 or 3 phosphodiesterases. Of course,background level inhibition of Type 1 or 3 phosphodiesterases ispermitted within the definition. Where the inhibitor inhibits Type 4 aswell as Type 1 and/or 3, but inhibits Type 4 to a greater extent (theamounts being subject to quantitative determination by assays describedherein), the phrase “preferentially inhibits Type 4 phosphodiesterases”is used herein (as distinct from “Type 4 specific.”

DESCRIPTION OF THE FIGURES

FIG. 1 is a gel showing the results of PCR on normal and CLL cells usingoligonucleotides specific for human phosphodiesterases.

FIG. 2 is a Northern blot using the PCR products of FIG. 1.

FIG. 3 graphically shows the PDE activity of CLL cells (left panel) anda murine B lymphoma cell line (right).

FIG. 4 graphically shows cAMP levels in CLL cells, WMC, and resting Bcells.

FIG. 5 shows the DNA fragmentation results on gel electrophoresis usinga 1.5% agarose gel wherein the bands are visualized with ethidiumbromide.

FIG. 6 shows the results of Hoechst 33342 flow cytometry.

FIG. 7 graphically shows the increasing percent of apoptotic cells as afunction of time and rolipram dose.

FIG. 8 graphically shows the different sensitivity of various cells tothe specific inhibitors of the present invention.

FIG. 9 graphically shows that rolipram blocks cAMP catabolism in bothsensitive and resistant lymphoid populations.

FIG. 10 graphically shows that sensitivity to rolipram mirrors thesensitivity to dbcAMP.

FIG. 11 graphically shows an increase in the percent apoptotic cellswith increasing doses of the inhibitor XX5.

FIG. 12 graphically shows that rolipram augments fludarabine-inducedapoptosis in CLL cells.

FIG. 13 graphically shows that rolipram augments chlorambucil-inducedapoptosis in CLL cells.

FIG. 14 graphically shows that rolipram augments doxorubicin-inducedapoptosis in CLL cells.

DESCRIPTION OF THE INVENTION

The present invention pertains to the treatment of patients with chroniclymphocytic leukemia with pharmaceutical compositions comprising Type 4cyclic adenosine monophosphate phosphodiesterase inhibitors, and moreparticularly, with an inhibitor that specifically inhibits Type 4 cyclicadenosine monophosphate phosphodiesterase. The description of theinvention discusses 1) apoptosis generally, 2) phosphodiesterases as atarget for CLL therapy, and 3) treating CLL patients.

A. Apoptosis and Intracellular cAMP Levels

The first observation that some lymphoid cells die following exposure toagents that raise intracellular cAMP levels was made by Daniel et al.who found that the murine lymphoma cell line S49.1 underwent cytolysisfollowing 48 hours of exposure to the combination of theophylline and acell permeable 3′:5′ cyclic AMP analog, dibutyryl cAMP (dbcAMP). V.Daniel et al., “Induction of cytolysis of cultured lymphoma cells byadenosine 3′:5′-cyclic monophosphate and isolation of resistantvariants,” Proc. Natl. Acad. Sci. USA 70:76 (1973). When mutant S49.1clones resistant to the cytolytic effects of dbcAMP were isolated insoft agar, they were shown to be defective in the regulatory subunit ofprotein kinase A, confirming that cytolysis occurred as a direct resultof PKA-mediated phosphorylation of unknown lymphoid target proteins.Subsequent work has demonstrated that cAMP-induced cytolysis occurs byapoptosis. D. J. McConkey et al., “Agents that elevate cAMP stimulateDNA fragmentation in thymocytes,” J. Immunol. 145:1227 (1990). Certainnormal T and B lymphoid subsets express the same marked sensitivity tocAMP-induced toxicity as tumor cell lines. Within the T lineage,CD4+CD8+ thymocytes appear to be more sensitive to the induction ofapoptosis by cAMP than mature T cells. Apoptosis in resting human Blymphocytes, which occurs spontaneously at a high rate in culture, canbe augmented by the addition of stimuli which elevate intracellular cAMPlevels, such as the diterpene adenylate cyclase activator forskolin. J.Lomo et al., “TGF-b1 and cyclic AMP promote apoptosis in resting human Blymphocytes,” J. Immunol. 154:1634 (1995).

B. Phosphodiesterases as a Target for Therapy of CLL

Cyclic AMP is catabolized within cells to 5′-AMP by 3′:5′ cAMPphosphodiesterases (PDE), a diverse group of enzymes encompassing 15gene products and 7 classes of enzymes which have proven to be thetarget of successful pharmaceutical agents for neurologic,cardiovascular and inflammatory disorders. Despite this large array ofcyclic nucleotide PDEs, only a subset of these enzymes have beenreported in human lymphoid cells. Among them, the most commonly reportedenzymes in human T cells are types 1, 3 and 4. Calcium-calmodulindependent type 1 PDE activity has been detected inphytohemagglutinin-stimulated but not resting peripheral bloodlymphocytes. One isoform from this family, PDE1B1, was recently detectedin acute lymphocytic leukemia cells; inhibition of this enzyme wasreported to induce apoptosis. PDE1 enzymes, which can catalyze thedegradation of both cAMP and cGMP, are specifically inhibited byvinpocetine (IC50=21 mMol/L). Two groups have reported both type 3 andtype 4 PDE in human T lymphocytes; lectin-mediated proliferation wascompletely suppressed only by treating cells with specific inhibitors ofboth classes of enzymes. While four human PDE4 genes have been cloned,only three of the isoforms (PDE4A, B and D) have been identified inlymphocytes. Type 4 enzymes are specifically inhibited by rolipram[4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone] (IC50=1 mMol/L) andthe structurally related compound XX5 (IC50=2 mMol/L). U. Schwabe etal., “4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711): apotent inhibitor of adenosine cyclic 3′,5′-monophosphatephosphodiesterases in homogenates and tissue slices from rat brain,”Molecular Pharmacology 12:900 (1976). H. Sheppard et al.,“Structure-activity relationships for inhibitors of phosphodiesterasefrom erythrocytes and other tissues,” Adv Cyclic Nucl Res 1:103 (1972).

Theophylline induces apoptosis in CLL cells, but it is not a specificType 4 inhibitor. Moreover, a clinical application of theophylline forCLL is complicated by its activity as an adenosine receptor antagonist.As an alternate approach, the present invention contemplates specificinhibitors.

C. Treating Patients

While the present invention is not limited by the nature of the priortreatment of the subject, it is contemplated that, in one embodiment,the present invention be utilized in patients who have not undergoneprior treatment for their condition (ie., naive patients), as well aspatients who have not responded to (or are refractory to) standardchemotherapeutic agents (e.g., alkylating agents). Thus, the presentinvention specifically contemplates treating patients who have failedstandard therapy.

On the other hand, the present invention specifically contemplates theuse of the inhibitors in combination with other drugs, including but notlimited to cytotoxic drugs. This combination therapy is based onfindings (described herein) that specific inhibitors can augmentapoptosis when used with such drugs.

It is contemplated that the methods of the present invention beadministered alone or can be administered with a pharmaceutical carrierselected on the basis of the chosen route of administration and standardpharmaceutical practice. In one preferred embodiment, the specificinhibitor is administered orally in solid dosage forms, such ascapsules, tablets, or powders, or in liquid dosage forms, such aselixirs, syrups, and suspensions; however, it can also be administeredparenterally, in sterile liquid dosage forms, or rectally in the form ofsuppositories.

One skilled in the art will be capable of adjusting the administereddose depending upon known factors such as the mode and route ofadministration; age, health, and weight of the recipient, nature andextent of symptoms, kind of concurrent treatment, frequency oftreatment, and the effect desired. In one embodiment, the dosage isincreased to overcome a non-responsive condition.

Additionally, the specific (and preferential) inhibitors of the presentinvention can be employed in admixture with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for parenteral (e.g., topical application) orenteral (e.g., oral) which do not deleteriously react with the activecompounds.

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils,benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such aslactose, amylose, or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, merely to name a few. Thepharmaceutical preparations can be sterilized and if desired mixed withauxiliary agents, e.g., lubricants, preservatives, stabilizers, wettingagents, emulsifier, salts for influencing osmotic pressure, buffers,coloring, flavoring, and/or aromatic substances and the like which do nodeleteriously react with the active compounds. They can also be combinedwhere desired with other agents, e.g., vitamins and/or antibiotics.

For enteral application, particularly suitable are tablets, liquids,drops, suppositories, or capsules. A syrup, elixir, or the like can beused wherein a sweetened vehicle is employed. Sustained or directedrelease compositions can be formulated, e.g., liposomes or those whereinthe active compound is protected with differentially degradable coating,e.g., by microencapsulation, multiple coatings, etc.

In this manner, the present invention may be introduced into a subjectin polymeric microspheres for the controlled release of the compound.Methods of producing microspheres from polymer can be found in U.S. Pat.No. 5,601,844 to Kagayama, et al., and U.S. Pat. Nos. 5,529,914 and5,573,934 to Hubbel, et al., herein incorporated by reference.

Other medicaments can be produced in a known manner, whereby the knownand customary pharmaceutical adjuvants as well as other customarycarrier and diluting agents can be used. Examples include, but are notlimited to, gelatins, natural sugars such as sucrose or lactose,lecithin, pectin, starch (for example cornstarch), alginic acid, tylose,talc, lycopodium, silica (for example colloidal silica), glucose,cellulose, cellulose derivatives for example, cellulose ethers in whichthe cellulose hydroxyl group are partially etherified with loweraliphatic alcohols and/or lower saturated oxyalchohols, for example,methyl hydroxypropyl cellulose, methyl cellulose, cellulose phthalate,stearates, e.g., methylstearate and glyceryl stearate, magnesium andcalcium salts of fatty acids with 12 to 22 carbon atoms, especiallysaturated acids (for example, calcium stearate, calcium laurate,magnesium oleate, calcium palmitate, calcium behenate and magnesiumstearate), emulsifiers, oils and fats, especially of plant origin (forexample, peanut oil, castor oil, olive oil, sesame oil, cottonseed oil,corn oil wheat germ oil, sunflower seed oil cod-liver oil), mono, di,and triglycerides of saturated fatty acids (C₁₂H₂₄O₂ to C₁₈H₃₆O₂ andtheir mixtures), e.g., glyceryl monostearate, glyceryl distearate,glyceryl tristearate, glyceryl trilaurate), pharmaceutically compatiblemono- or polyvalent alcohols and polyglycols such as glycerine,mannitol, sorbitol, pentaerythritol, ethyl alcohol, diethylene glycol,triethylene glycol, ethylene glycol, propylene glycol, dipropyleneglycol, polyethylene glycol 400, and other polyethylene glycols, as wellas derivatives of such alcohols and polyglycols, esters of saturated andunsaturated fatty acids (2 to 22 carbon atoms, especially 10 to 18carbon atoms), with monohydricaliphatic alcohols (1 to 20 carbon atomalkanols), or polyhydric alcohols such as glycols, glycerine, diethyleneglycol, pentaerythritol, sorbitol, mannitol, ethyl alcohol, butylalcohol, octadecyl alcohol, etc., e.g., glyceryl stearate, glycerylpalmitate, glycol distearate, glycol dilaurate, glycol diacetate,monoacetin, triacetin, glyceryl oleate, ethylene glycol stearate; suchesters of polyvalent alcohols can in a given case be etherified, benzylbenzoate, dioxolane, glycerine formal, tetrahydrofurfuryl alcohol,polyglycol ethers with 1 to 12 carbon atom alcohols, dimethyl acetamide,lactamide, lactates, e.g., ethyl lactate, ethyl carbonate, silicones(especially middle viscosity dimethyl polysiloxane).

For injectable solutions or suspensions, non-toxic parenterallycompatible diluting agents or solvents can be used, for example: Water,1,3 butane diol, ethanol, 1,2-propylene glycol, polyglycols in a mixturewith water, Ringer's solution, isotonic solution of sodium chloride oralso hardened oils including synthetic mono or diglycerides or fattyacids like oleic acid.

Known and customary solution assistants or emulsifiers can be used inthe production of the preparations. The following are examples ofsolution assistants and emulsifiers which can be used:Polyvinylpyrrolidone, sorbitan fatty acid esters such as sorbiantrioleate, phosphatides such as lecithin, acacia, tragacath,polyoxethylated sorbitan monooleate and other ethoxyated fatty acidesters of sorbitan, polyoxyethylated fats, polyoxyethylatedoleotriglycerides, linolized oleotriglycerides, polyethylene oxidecondensation products of fatty alcohols, alkyl phenolene or fatty acidsor also 1-methyl-3-(2-hydroxyethyl)imidazolidone-(2). The termpolyoxyethylated means in this context that the substances in questioncontain polyoxyethylene chains whose polymerization is generally between2 to 40 and especially between 10 to 20.

Furthermore, there can be added preservatives stabilizers, buffers, forexample, calcium hydrogen phosphate, colloidal aluminum hydroxide, tastecorrectives, antioxidants and complex formers (for example, ethylenediamine tetraacetic acid) and the like. In a given case forstabilization of the active molecule, the pH is adjusted to about 3 to 7with physiologically compatible acids or buffers. Generally, there ispreferred as neutral as possible to weak acid (to pH 5) pH value.

As antioxidants, there can be used, for example, sodiummeta bisulfite,ascorbic acid, gallic acid, alkyl gallates, e.g., methyl gallate andethyl gallate, butyl hydroxyanisole, nordihydroguararetic acid,tocopherols as well as tocopherol and synergists (materials which bindheavy metals by complex formation, for example, lecithin, ascorbic acid,phosphoric acid). The addition of synergists increases considerably theantioxidant activity of tocopherol. As preservatives, there can be used,for example, sorbic acid, p-hydroxybenzoic acid esters (for example,lower alkyl esters such as the methyl ester and the ethyl ester) benzoicacid, sodium benzoate, trichloroisobutyl alcohol, phenol, cresol,benzethonium chloride, and formalin derivatives.

EXPERIMENTAL

The following example serves to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

A. Reagents

The following reagents were obtained from commercial sources: alkalinephosphatase, cAMP, dibutyryl cAMP, calmodulin, forskolin (Sigma ChemicalCo., St Louis, Mo.); vinpocetine (Alexis Biochemicals, San Diego,Calif.); recombinant human IL-2 (Genzyme, Boston, Mass.); F(ab′)2fragment goat anti-human IgG and IgM (Jackson ImmunoresearchLaboratories, West Grove Pa.); Hoechst 33342 (Molecular Probes, Eugene,Oreg.). Rolipram (racemate of4-[3′-cyclopentyloxy-4′-methoxyphenyl]-2-pyrrolidone) was a gift fromDr. Ronald Wohl, Berlex Laboratories (Wayne, N.J.).

B. Patient Selection

After IRB-approved informed consent, blood was drawn in heparinizedtubes from patients with flow cytometry-verified CLL who were eitheruntreated or at least one month post-chemotherapy. Patients with activeinfections or other serious medical conditions were not included in thisstudy. Charts were reviewed to establish patients'sensitivity tochemotherapy and the stage of CLL. Resistance to a chemotherapeuticagent was defined as a rise in peripheral leukemic cell count orprogression of adenopathy or splenomegaly prior to the initiation of thenext scheduled cycle of chemotherapy.

C. Cell Purification and Culture

Mononuclear cells were obtained by density gradient centrifugation overHistopaque 1077 (Sigma Chemical Company, St. Louis, Mo.). As flowcytometry demonstrated that CLL cells made up more than 95% of themononuclear cells so purified, both apoptosis and cAMP assays wereperformed with these patient cell preparations. For PCR experiments onCLL cells or for all experiments on normal circulating B cells, thewhole mononuclear cells were further purified by incubation with Dynalanti-CD19 magnetic beads at a 1:1 bead:cell ratio, extensive washingwith a magnetic particle concentrater and elution with CD19 Detachabeadreagent (Dynal, Lake Success, N.Y.). Leukemic cells from two CLLpatients showed sensitivity to rolipram-mediated apoptosis whether ornot they were firther purified by anti-CD19 magnetic beads. Cells werecultured in RPMI 1640 media (Biowhittaker, Walkersville, Md.)supplemented with 10% fetal calf serum, 50 uMol/L 2-mercaptoethanol, 2mnMol/L L-glutamine, 10 mMol/L Hepes pH 7.4, 100 U/mL penicillin, and100 U/mL streptomycin (Sigma Chemical Company, St. Louis, Mo.) at 37° C.and 5% CO₂ in air.

D. RT PCR and Northern Analysis

RNA was isolated from CLL or whole mononuclear cells using Ultraspecreagent (Biotecx, Houston, Tex.). cDNA was synthesized from 10 ug oftotal RNA using oligo d(T) primers and Maloney murine leukemia virusreverse transcriptase in a final volume of 40 uL (Stratagene, La Jolla,Calif.). One mL of the first strand cDNA product was then used astemplate for PCR amplification with AmpliTaq DNA polymerase (RocheMolecular Systems, Branchburg, N.J.) by 40 thermocycles of 94° C. for 1minute, 600° C. for 1 minute and 72° C. for 1 minute. The PDE PCR assayproducts were as follows with oligonucleotide sequences given 5′->3′:Human PDE1B1 (Genbank accession # U56976) was 430 bp (1st base 1660;sense=GTC TTC ATT GAG TCC AAA GTG, antisense=GAC CTG CCA GCT AAG ATCTGG). Human PDE3A (cGIP1, HSPDE3B) (X95520) was 340 bp (1st base 2999,sense=GTA ACT CCT ATG ATG CTG CTG G, antisense=CTA TTC CTC TTC ATC TGCCTC). Of note, these PDE3 PCR oligonucleotides are selective for thehuman cGIP1 PDE, homologous to rat PDE3A, as the amplified sequence hasonly 50% nucleotide homology to the cardiac/platelet form of human PDE3(cGIP2). Human PDE4A (M37744) was 461 bp (1st bp 1819, sense=GGA GGA AGAAAT ATC AAT GGC CC, antisense=GAT GTG TCC TCC CCA AAT GTC). Human PDE4B(L20966) was 479 bp (1st bp 2213, sense=ATT CTG AAG GAC CTG AGA AGG,antisense=CAG TGA GTT CAG TCA CTG TCG). For hybridization to Northernblots, these PCR products were subcloned into a plasmid vector (pCRII,Invitrogen, Carlsbad, Calif.) and subsequently utilized for PCR-basedamplification of a32P dATP-labelled probes.

E. cAMP Assay

One million cells in 1 mL were treated for two hours with or withoutdrugs. 0.8 mL of cells were pelleted by spinning at 4,000 rpm(RCF=1,310) in microcentrifuge tube. After discarding 0.7 mL, 400 uL ofethanol was added, vortexed and left on ice for five minutes.Particulate cell debris was removed by centrifugation at 14,000 rpm(RCF=16,000). The supernatant was stored at −20° C. until the day ofassay, at which time it was dried in a Speedivac (Savant, Farmingdale,N.Y.) to a volume of 50 uL. After ten-fold dilution in 10 mMol/L Tris pH8.0, 1 mMol/L EDTA, the cAMP sample was analyzed for cAMP concentrationusing a cAMP RIA kit (NEN, Boston, Mass.) according to themanufacturer's instructions using the nonacetylated protocol.

F. cAMP PDE Assay

The technique of Robiesek et al., itself adapted from an assay describedby Thompson and Appleman, was used in modified form. S. A. Robicsek etal., “Multiple high-affinity cAMP-phosphodiesterases in humanT-lymphocytes,” Biochem. Pharmacol. 42:869 (1991). W. J. Thompson and M.M. Appleman, “Multiple cyclic nucleotide phosphodiesterase activitiesfrom rat brain,” Biochemistry 10:311 (1991). 150 million purified CLLcells were pelleted and sonicated (Branson 350 Sonifier with micropitprobe, output =2, 50% duty cycle) on ice in 1.0 mL of a buffer whichcontained 20 mMol/L Tris (pH 6.8), 1 mMol/L EDTA, aprotinin (50 u/mL),pepstatin (1 mg/mL), PMSF (1 mMol/L) and 3.75 mMol/L b-ME. Assay buffercontained 100 mMol/L Tris (pH 8.0), 20 mMol/l MgCl₂, 0.2% BSA and 7.5mMol/L b-ME. [³H]-cAMP (NEN, Boston, Mass.) was incubated with PDE for10 minutes at 30° C. in 20 uL volumes (10 uL of sonication buffer and 10uL of assay buffer) which contained 0.22 units of alkaline phosphatase.10 uMol/L rolipram or 0.2 mMol/L calcium/20 nMol/L calmodulin were addedto the assay buffer as appropriate. Reactions were halted by theaddition of 0.5 mL of a 1:3 slurry w/v slurry of AG1-X8 anion exchangeresin and a mixture of equal volumes of water and isopropanol. The resinbound the unreacted nucleotide but not the dephosphorylated nucleoside.Microcentrifuge tubes were spun at 3000 rpm (RCF=735) for 15 minutes.The radiolabelled nucleosides in the supernatant were counted usingEcoscint scintillation fluid (National Diagnostics, Atlanta, Ga.). Threeto five enzyme dilutions were assayed to determine each velocity.Linearity of velocity with respect to enzyme concentration and time wereverified.

G. DNA Ladder Gel Assay

10 million purified CLL cells were harvested by centrifugation followingexposure to drugs during a 72 hour tissue culture incubation. Cells werelysed in 0.5 mL of 20 mMol/L Tris (pH 7.4), 0.4 mMol/L EDTA, 0.25%Triton X. After 15 minutes of incubation at room temperature, nucleiwere removed by centrifugation at 14,000 rpm (RCF=16,000). Thesupernatant was transferred to a new tube and soluble DNA precipitatedovernight at −20° C. following the addition of 55 mL of 5 Mol/L NaCl and550 mL of isopropanol. After centrifugation at 14000 rpm for 10 minutes,followed by a 70% ethanol wash, the pellet was resuspended in 20 uL of10 mMol/L Tris (pH 8.0), 1 mMol/L EDTA, 0.1 mg/mL RNase and incubated at37° C. for 30 minutes prior to electrophoresis on 1.6% TBE agarose gels.DNA fragments were visualized by UV light after staining the gels withethidium bromide.

H. Hoechst 33342 Apoptosis Assay

Hoechst 33342 was dissolved in water and frozen at 33 mg/mL at −20° C.One million cells/well were incubated in duplicate or triplicate in 48well tissue culture plates (Costar, Cambridge, Mass.) with or withoutdrug treatment for 48 hours in 1 mL of culture media. Cells weretransferred to 12×75 mm polystyrene Falcon® 2054 FACS tubes (BectonDickinson Labware, Lincoln Park, N.J.) and incubated for ten minutes at37° C. with Hoechst 33342 at a final concentration of 0.25 mg/mL. 23Cells were stored on ice until analysis on a FACS Vantage flow cytometer(Becton Dickinson, San Jose, Calif.). Hoechst 33342 dye fluorescence wasexcited with a UV laser and detected using a 450 bandpass filter; Datawas analyzed using Cellquest software (Becton Dickinson, San Jose,Calif.).

EXAMPLE 1

This example describes the identification of PDE targets in CLL cells.As an initial approach to identify potential PDE targets in CLL, PCR wasperformed on cDNA derived from unpurified mononuclear cells of a CLLpatient with oligonucleotides specific for human PDB1-1B, PDE3A, PDE-4Aand PDE-4B, all of which have been previously identified in variousnormal and malignant primary lymphoid cells. Appropriate sized PCRproducts were detected from all four transcripts using this template. Inorder to reduce the likelihood of amplifying non-leukemic celltranscripts, we purified CD19-positive cells from this and two other CLLpatients prior to synthesizing cDNA. PDE1-1B, PDE-4A and PDE-4B werestill detected in these three templates, as they were in cDNA derivedfrom normal whole mononuclear cells FIG. 1 shows that CLL cells containtranscripts for PDE 1B1, PDE4A and PDE4B. The cDNA utilized in the PCRin the bottom 3 panels was derived from leukemic cells from threedifferent patients purified by positive selection for CD19 expression.The lowest band in the MWM lane on the left in the upper panel is 603bp. Expected PCR product sizes for PDE1, 3A, 4A and 4B are 430, 340, 461and 470 bp respectively.

Using the same four PCR products as probes on Northern blots, onlyPDE-4B transcript was detectable in 10 ug of loaded RNA. FIG. 2 showsthat PDE4B levels fall in CLL cells following culture but are partiallymaintained by treatment with 10 uMol/L rolipram. RNA was isolated from20 million CLL cells immediately after cell purification (CT), or after6 hours culture in media alone (6 Hr) or with addition of 10 uMol/Lrolipram (Roli). Equal loading and transfer of RNA was confirmed byhybridization with an actin probe as shown. These results arerepresentative of Northern analysis performed on leukemic cells from twopatients. PDE-4B transcript levels were significantly higher in freshlyisolated CLL cells than in CLL cells which had been cultured for sixhours. Addition of 10 uMol/L rolipram, a type 4 PDE-specificphosphodiesterase inhibitor, significantly reduced this fall in PDE-4Btranscript levels during the six hour culture period.

EXAMPLE 2

In this example, it was deterniined whether the above-described PDBtranscripts were translated into protein with constitutive activity. PDEenzyme assays were performed on CLL cell lysates. FIG. 3 showsLineweaver-Burk analysis of PDE enzymatic activity in lysates of CLLcells and Bal-17 cells (the CLL data are representative of enzymaticassays on three patients). Clearly, Type 1 and 4 PDE activity differsbetween a murine B lymphoma cell line (Bal-17) and CLL cells. We foundno evidence of type 1 PDE activity in CLL cells as the basal PDEactivity was not augmented by the addition of calcium and calmodulin(FIG. 3, left). PDE activity in CLL cells was inhibited by 10 uMol/Lrolipram (rolipram altered both K_(m) and the V_(max), consistent withits known activity as a noncompetitive antagonist) but was not augmentedby the addition of 0.2 mMol/L calcium and 20 nMol/L calmodulin. On theother hand, Bal-17 PDE activity is augmented by the addition of calciumand calmodulin (FIG. 3, right). That is to say, it was possible toidentify substantial constitutive type 1 PDE activity in the murine Blymphoma cell line Bal-17, as demonstrated by a rise in PDE activitywith the addition of calcium and calmodulin.

EXAMPLE 3

In this example, it was determined what role type 1 and type 4 PDEactivity might play in the catabolism of cyclic nucleotides in CLLcells. cAMP levels were measured after culturing leukemic cells withvarying concentrations of PDE-isoform specific inhibitors, either aloneor in conjunction with an activator of adenylate cyclase, the diterpeneforskolin. Specifically, one million freshly isolated cells wereincubated with the indicated drugs for two hours prior to analysis ofcAMP in cell lysates using a radioimmunoassay. The results are shown inFIG. 4 and the data are the mean of three individually assayed culturewells. The experimental conditions indicated with an asterisk had agreater [cAMP] than, as appropriate, the untreated or forskolin-treatedcontrol cells (t test: one-tailed significance level <0.05).

In panel D, vinpocetine was added at 30 uMol/L. When incubated withforskolin and the PDE-1 inhibitor vinpocetine, CLL cell cAMP was notaugmented above levels induced by forskolin alone (FIG. 4, panel D).Incubation of Bal-17 cells with vinpocetine reduced both basal andforskolin-stimulated cAMP levels, a result in keeping with the reportedprimary effect of this drug on cGMP rather than cAMP levels (data notshown). In contrast, addition of the type 4 PDE inhibitor rolipramaugmented CLL cAMP levels, both when used alone and more dramaticallywhen combined with forskolin (FIG. 4, Panels A-D). CLL cells were notunique with respect to their response to these isoform-specificinhibitors. cAMP levels in both a predominantly T cell population (IL-2supplemented normal whole mononuclear cells; >90% CD3+ T cells by flowcytometry) and magnetic-bead purified CD19+ B cells rose followinginhibition of type 4 but not type 1 PDE activity (FIG. 4, Panels E and Fand data not shown).

EXAMPLE 4

This example demonstrates that Type 4 PDE inhibition induces CLLapoptosis The above-described results identified the type 4 cAMPphosphodiesterase family as an important regulator of cAMP levels in CLLcells. Consequently, a study of the activity of type 4-specific PDEinhibitors as inducers of cAMP-mediated apoptosis in leukemic cells fromCLL patients was carried out. CLL cells were incubated for 72 hourseither alone or with 10 uMol/L rolipram, 40 uMol/L forskolin or bothagents. We tested whether rolipram induces intemucleosomal DNAfragmentation characteristic of apoptosis by isolating soluble DNA fromthe leukemic cells with detergent treatment, then removing DNA fromintact non-apoptotic nuclei by centrifugation. Specifically, soluble DNAwas isolated from ten million CLL cells cultured for 72 hours in media(CT), 10 uMol/L rolipram (Roli), 40 uMol/L forskolin (Fsk) or acombination of the latter two agents (Ro/Fs). DNA fragments wereresolved by electrophoresis on a 1.5% agarose gel and visulized withethidium bromide. FIG. 5 shows the results. These data arerepresentative of the four leukemic cell samples tested. As shown inFIG. 5, while culture of CLL cells in media alone resulted in only afaint DNA “ladder”, treatment with rolipram and/or forskolin resulted inpronounced internucleosomal DNA fragmentation.

As a more quantitative analysis of CLL apoptosis, we utilized a flowcytometry method in which apoptotic cells are distinguished both bytheir reduced size (FSC) and their increased uptake of the lipophilic UVfluorescent dye Hoechst 33342 (FL-4) when the intact, heterogeneous cellpopulation is incubated with a low concentration of the dye (0.25 ug/mL)for 10 minutes at 37° C. Specifically, cells were cultured for 72 hoursin media (1), 1uMol/L rolipram (2), 40 uMol/L forskolin (3) or acombination of the two drugs (FIG. 6). The abcissa reflects forwardlight scatter and the ordinate Hoechst 33342 fluorescence. Apoptoticcells are characterized by reduced forward light scatter and increasedHoechst 33342 fluorescence. Previous reports of cAMP induced lymphoidapoptosis have noted that this form of programmed cell death may take 48to 72 hours to develop maximally. Using the Hoechst 33342 assay in atime course experiment, we found that the combination of 10 uMol/Lrolipram and 40 uMol/L forskolin induced significant CLL apoptosis whichplateaued 48 to 72 hours after the addition of these drugs (FIG. 7, leftpanel). CLL cells were cultured for 72 hours in one mL of media with theindicated concentration of rolipram with (black bars) or without(stippled bars) the addition of 40 uMol/L forskolin. Using the 72 hourculture period, we found a dose dependent increase in CLL cell apoptosiswhen leukemic cells were incubated with rolipram (FIG. 7, right panel).Treatment of CLL cells with forskolin alone induced moderate apoptosis,but combination of forskolin with even low doses of rolipram resulted ina supra-additive effect on induction of CLL apoptosis (FIG. 7, rightpanel). Similar results were obtained with a structurally distinct PDE4inhibitor, XX5, or when isoproterenol or prostaglandin E2 were utilizedto activate CLL adenylate cyclase rather than forskolin (data notshown).

When CLL cells were incubated with the type 1 PDE inhibitor,vinpocetine, they underwent apoptosis at dosages of 10 or 30 uMol/L butnot at 2 uMol/L. Given that vinpocetine failed to augment cAMPaccumulation and that we were unable to detect type 1 PDE activity inCLL cells, we suspect that this drug may induce apoptosis by a mechanismunrelated to cAMP. Consistent with this hypothesis, the kinetics of CLLapoptosis were different when vinpocetine was utilized with peakapoptosis by 24 rather than 48 hours (data not shown). Nonetheless, wecannot rule out either a temporally restricted or a topologicallycompartmentalized cAMP-mediated apoptotic effect from this drug.

Given that CLL is a clinically heterogeneous disease, we tested a totalof 14 CLL patients of varying clinical stage, treatment history andknown resistance to chemotherapeutic agents for the sensitivity of theircells to phosphodiesterase inhibitor-mediated apoptosis. Patients wereassessed for the apoptotic sensitivity of their leukemic cells torolipram, forskolin or both drugs. In samples from ten“rolipram-sensitive patients”, treatment with 10 uMol/L rolipram inducedapoptosis in more than a third of the leukemic cells, with overallapoptosis ranging from 44 to 80% (see Table 1 for tabulated results).Among the seven rolipram-sensitive patients whose cells were treatedwith both conditions, 40 uMol/L forskolin as a single agent induced lessapoptosis than rolipram alone, suggesting that blockade of cAMPcatabolism induced a more potent apoptotic signal than furtheraugmentation of adenylate cyclase activity (p<0.08, Wilcoxonsigned-ranks test for matched pairs). Among four relativelyrolipram-resistant patient samples (Pt #'s 11-14: Table 1), the absoluteincrease in apoptotic cells was less than 33%, with overall apoptosisranging from 14 to 40%. Nonetheless, addition of forskolin to rolipramaugmented apoptosis (68% and 69%) in two of the three patient samplesexamined within this group.

TABLE 1 Pt. Rai Drug Res. Basal Roli Fsk R/Fs dbcAMP 1. III — 47 80 NDND ND 2. IV Ch, Cy 18 ± 2 79 46 89 ND 3. IV Ch, CH 38 77 ± 1 59 ± 0 79 ±1 71 ± 3 4. I — 31 ± 2 76 ± 2 ND ND ND 5. IV — 18 57 ± 2 ND ND ND 6. IVCh 17 ± 4 54 ± 1 30 ± 4 60 ± 2 39 ± 4 7. II Ch 13 ± 2 47 ± 2 32 ± 0 58 ±0 32 ± 4 8. 0 —  5 46 22  55* ND 9. IV Ch, Cy, Fl   4 ± 0 44 ± 3 17 ± 477 ± 2 46 ± 1 10. I — 11 44 ± 0 26 ± 4 56 ± 0 ND 11. 0 — 24 ± 4 40 ± 040 ± 3 69 ± 0 39 ± 2 12. IV Ch, Fl, 2C   0 ± 4 38 ± 1 43 ± 1 68 ± 0 ND13. 0 —   2 ± 0 34 ± 0 ND ND 23 ± 6 14.** I — 13 ± 0 14 ± 0 19 ± 3 37 ±3 22 ± 0 Apoptosis of CLL patients' leukemic cells following treatmentwith 10 uMol/L rolipram, 40 uMol/L forskolin or 100 uMol/L dbcAMP.Cultures were performed for three days and the percentage of apoptoticcells measured by Hoechst 3342 flow cytometry. Drugs: Ch = chlorambucil,CH = CHOP, Cy = cyclophosphamide, Fl = fludarabine, 2C =2-chlorodeoxyadenosine. SEMs are shown for samples done in triplicate;the remainder of the values are the mean of duplicate cultures.*Rolipram was at 1 uMol/L. **This patient had a significant populationof cells with prolymphocyte morphology.

EXAMPLE 5

This example describes the effect of PDE4 inhibition on normal B and Tcells. Given that cAMP has been reported to be cytocidal to specificnormal lymphocyte subsets, it was determined whether rolipram alsoinduced apoptosis in normal circulating human B and T cell populations.Specifically, one million WMC were cultured with the indicatedconcentration of rolipram with or without the addition of 40 uMol/Lforskolin for 72 hours in the presence of 2 units/mL IL-2. Apoptosis wasdetermined by Hoechst 33342 flow cytometry. It was found that IL-2cultured WMC (>90% CD3+ T cells by flow cytometry) were resistant toeven high doses of rolipram and forskolin (FIG. 8, upper panel). Incontrast, magnetic bead purified CD19+B cells were sensitive torolipram, although the increment in apoptosis observed was superimposedon a high basal apoptosis rate that has previously been reported incultured human B cells. Given that crosslinking of cell surfaceimmunoglobulin on resting B cells has been reported to reduce basal andforskolin-induced apoptosis in culture, we also stimulated CD19+ cellswith a polyclonal Fab′2 anti-IgM/IgG reagent 30 minutes prior to theaddition of the phosphodiesterase inhibitor. Prior stimulation throughsurface Ig markedly reduced both basal apoptosis and the sensitivity ofthe B cells to rolipram (FIG. 8, middle panel). In contrast, anti-Igstimulation of CLL cells derived from two patients failed to protectthese cells from rolipram-induced apoptosis, a result that is consistentwith reported defects in CLL cells in either surface m heavy chainexpression or mutations in the B29 (CD79b) B cell receptor accessoryprotein (FIG. 8, bottom panel).

The alteration in rolipram sensitivity in anti-Ig stimulated B cells wasnot due to a change in the ability of this drug to augment cAMP levelsat two hours in these cells, as rolipram raised cAMP levels equivalentlyin unstimulated or stimulated CD19+ B cells (FIG. 9). In order todetermine whether the rolipram-sensitive cell populations had a moreprolonged elevation of cAMP than the insensitive cell populationsfollowing drug treatment, we measured cAMP levels 6 or 24 hours afteraddition of rolipram, times at which levels of apoptosis were still loweven in sensitive populations. For each of the four cell populations,two or six hours after drug treatment, cAMP levels were higher forrolipram/forskolin-treated cells than for forskolin only treated cells(test: one tailed significance level<0.05) (FIG. 9). By 24 hours, therewas no longer significant rolipram-induced augmentation offorskolin-stimulated cAMP accumulation in any of the four cellpopulations (FIG. 9). Thus, the degree of cAMP augmentation by rolipramdid not predict the sensitivity of cell populations to induction ofapoptosis by this drug at any time point tested.

EXAMPLE 6

In this example, the sensitivity of four populations to the cellpermeable cAMP analog, dibutyryl cAMP was examined. Specifically, 1million CLL cells, resting or anti-Ig activated CD19+ cells, or IL-2supplemented WMC were cultured for 72 hours with 10 uMol/L rolipram, 40uMol/L forskolin or the dbcAMP concentration indicated in FIG. 10 (inuMol/L) prior to measurement of apoptosis by Hoechst 33342 flowcytometry. The SEMs of tiplicate cultures are indicated. As shown inFIG. 10, a strong correlation was found between rolipram and dbcAMPinduced apoptosis. For CLL cells and resting B cells, the percentage ofapoptotic cells increased significantly relative to control cells aftertreatment with rolipram and forskolin or concentrations of dbcAMPgreater than or equal to 30 mMol/L (t test: one tailed significancelevel <0.05). For anti-Ig stimulated B cells or IL-2 cultured WMC,comparable treatments did not significantly increase the percentage ofapoptotic cells. Consistent with these results, in the seven CLLpatients studied thus far, there has also been a correlation betweensensitivity to rolipram and sensitivity to dbcAMP (see Table 1). Thesedata indicate that type 4 PDE is the relevant target for rolipram in itsinduction of apoptosis in CLL cells.

EXAMPLE 7

It is not intended that the present invention be limited to only oneparticular inhibitor. The present invention contemplates the treatmentof patients with chronic lymphocytic leukemia (CLL) with a variety ofinhibitors that specifically inhibit Type 4 cyclic adenosinemonophosphate phosphodiesterase. For example, FIG. 11 graphically showsan increase in the percent apoptotic cells with increasing doses of theinhibitor XX5. One million purified CLL cells were cultured for threedays in media (control), 40 uM forskolin (F) and/or the indicatedconcentrations of the PDE4 inhibitor XX5. Cells were then harvested andanalyzed for apoptosis using the Hoechst 33342 FACS assay. SEM oftriplicate cultures is shown.

EXAMPLE 8

In this example, it is demonstrated that the specific inhibitors of thepresent invention augment apoptosis induced by commonly used drugs(e.g., doxorubicin, chlorambucil and fludarabine). FIG. 12 graphicallyshows that rolipram augments fludarabine-induced apoptosis in CLL cells.One million CLL cells were incubated for three days in the presence ofmedia (control), theophylline (50 ug/mL), forskolin (40 uMol/L),rolipram (10 uMol/L), fludarabine (0.3-3.0 uMol/L) or combinations ofthese drugs. The percentage of apoptotic cells was determined by Hoechst33342 FACS analysis. SEM of triplicate cultures is shown. The right andleft graphs represent data derived from the cells of two different CLLpatients.

FIG. 13 graphically shows that rolipram augments chlorambucil-inducedapoptosis in CLL cells. CLL cells were incubated for three days withmedia (control), chlorambucil (indicated concentration in uMol/L),rolipram (10 uMol/L) or a combination of chlorambucil and rolipram. Thepercentage of apoptotic cells was then determined by Hoechst 33342 FACSanalysis. SEM of triplicate samples are shown.

FIG. 14 graphically shows that rolipram augments doxorubicin-inducedapoptosis in CLL cells. One million CLL cells were cultured for threedays with theophylline (50 ug/mL), rolipram (10 uMol/L), forskolin (F:40 uMol/L), doxorubicin (0.03 or 0.1 uMol/L) or combinations of thesedrugs as indicated. The percentage of apoptotic cells was determined byHoechst 33342 FACS analysis. SEM of triplicate cultures is shown.Consequently, the present invention specifically contemplates the use ofthe inhibitors in combination with other drugs, including but notlimited to cytotoxic drugs.

From the above, it should be clear that, inhibition of type 4 cAMPphosphodiesterase activity is a novel means by which to triggerapoptosis in chronic lymphocytic leukemia cells in vitro. Treatment ofCLL cells with the PDE4 family-specific inhibitor rolipram raised cAMPlevels and induced apoptosis in a dose and time-dependent manner, aneffect which correlated with apoptosis induced by dbcAMP. Treatment ispossible even where patients demonstrated clinical resistance tochlorambucil, cyclophosphamide and/or fludarabine (see Table 1).

If PDEs are to be a useful pharmacologic target in the therapy of CLL,drug dosages which trigger apoptosis in leukemic cells in vivo must haveclinically tolerable effects on other tissues. Despite their widespreadtissue distribution, type 4 inhibitors have been used effectively asanti-inflammatory drugs in animal models of asthma, inhibiting pulmonaryeosinophil accumulation after allergen challenge of sensitized animals.Rolipram has been studied extensively as an antidepressant in humans andis well tolerated, although higher dosages are emetogenic.

An effort has been made here to determine whether rolipram's ability toinduce apoptosis in the leukemic cells of some CLL patients is unique tothis malignant population or shared by normal circulating lymphocytes.We observed that IL-2 cultured WMC and sIg-triggered B cells werelargely insensitive to both rolipram and dbcAMP-induced apoptosis, whiletreatment of non-stimulated B cells with rolipram or dbcAMP induced amoderate increase in apoptosis, albeit superimposed on considerablebasal apoptosis.

BCL-2 and related proteins are likely to regulate sensitivity toapoptosis in CLL and are also potential targets for cAMP-mediatedsignal-transduction. Although less than 10% of CLL patients havechromosomal translocations involving BCL-2, hypomethylation and highlevel BCL-2 transcription is common.

I claim:
 1. A method, comprising: a) providing: i) a patient having ofchronic lymphocytic leukemia, and ii) a formulation comprising rolipram;and b) administering a therapeutically effective dose of saidformulation to said patient under conditions such that said leukemia isreduced.
 2. The method of claim 1, wherein said administering is enteraladministration.
 3. The method of claim 2, wherein said enteraladministration is oral administration.
 4. The method of claim 1, whereinsaid administering is parenteral administration.
 5. The method of claim1, wherein said patient is a naive patient.
 6. The method of claim 1,wherein said patient is immunocompromised.
 7. The method of claim 1,wherein said patient is unresponsive to chemotherapy winth alkylatingagents.